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AUGUST: WEEK 1
August, 1stTwo colonies for E28-AGAIN and E41-AGAIN plates were picked and inoculated in LB + Cm12.5; J101-31, J101-E5 and J101-E7 plasmids were extracted:
These plasmids were digested with EcoRI and SpeI restriction enzymes and screened by gel-electrophoresis; a medium-size agarose gel was then prepared.
Insert lengths were all correct so J101-31, J101-E5, J101-E7 were transformed from MiniPrepped DNA in MGZ1 competent cells. E37-2, E38-1, E39-1, E40-2 and E42-1 were inoculated in 5 ml LB + Cm12.5 and grown ON for plasmid extraction. August, 2ndMGZ1 J101-31, J101-E5 and J101-E7 plates were all grown; one colony was picked from each of them and inoculated in LB + Cm12.5. Plasmids harboring E28-AGAIN-1, E28-AGAIN-2, E41-AGAIN-1, E28-AGAIN-2, E37-2, E38-1, E39-1, E40-2 and E42-1 were extracted with MiniPrep kit from ON saturated cultures; purified DNA was quantified with NanoDrop Spectrophotometer:
Digestions of E28AGAIN-1, E28AGAIN-2, E41AGAIN-1 and E42AGAIN-2 were performed for screening; a 5x mix was prepared and for each sample the volumes specified in the table below were used:
A small-size agarose gel was prepared for gel electrophoresis.
Unfortunately insert length was correct only for E28-AGAIN-1 so we ligated again E41:
Glycerol stocks were prepared according to the protocol for J101-31, J101-E5 and J101-E7.
August, 3rd
T9002-ENTERO4C5 plate was grown and a colony was picked and inoculated in LB + Amp + Cm12.5; E41N ligation was transformed in 100 μl of MGZ1 competent cells and plated on a LB + Cm12.5 plate.
Then samples were centrifuged at 13.000 rpm; finally the supernatant was collected and stored at -20°C.
August, 4th
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Team:UNIPV-Pavia/Calendar/August/week1
From 2011.igem.org
(Difference between revisions)
Line 18: | Line 18: | ||
<center> | <center> | ||
- | <table | + | <table class="data"> |
<tr> | <tr> | ||
- | <td><b>Plasmid</b></td> | + | <td class="row"><b>Plasmid</b></td> |
- | <td><b>DNA (ng/μl)</b></td> | + | <td class="row"><b>DNA (ng/μl)</b></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td>J101-31</td> | + | <td class="row">J101-31</td> |
- | <td>???</td> | + | <td class="row">???</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td>J101-E5</td> | + | <td class="row">J101-E5</td> |
- | <td>???</td> | + | <td class="row">???</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td>J101-E7</td> | + | <td class="row">J101-E7</td> |
- | <td>???</td> | + | <td class="row">???</td> |
</tr> | </tr> | ||
Line 70: | Line 70: | ||
<center> | <center> | ||
- | <table | + | <table class="data"> |
<tr> | <tr> | ||
- | <td><b>Plasmid</b></td> | + | <td class="row"><b>Plasmid</b></td> |
- | <td><b>DNA (ng/μl)</b></td> | + | <td class="row"><b>DNA (ng/μl)</b></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td>E28AGAIN-1</td> | + | <td class="row">E28AGAIN-1</td> |
- | <td>21.4</td> | + | <td class="row">21.4</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td>E28AGAIN-2</td> | + | <td class="row">E28AGAIN-2</td> |
- | <td>21.0</td> | + | <td class="row">21.0</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td>E41AGAIN-1</td> | + | <td class="row">E41AGAIN-1</td> |
- | <td>33.7</td> | + | <td class="row">33.7</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td>E41AGAIN-2</td> | + | <td class="row">E41AGAIN-2</td> |
- | <td>27.7</td> | + | <td class="row">27.7</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td>E37-2</td> | + | <td class="row">E37-2</td> |
- | <td>18.5</td> | + | <td class="row">18.5</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td>E38-1</td> | + | <td class="row">E38-1</td> |
- | <td>26.0</td> | + | <td class="row">26.0</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td>E39-1</td> | + | <td class="row">E39-1</td> |
- | <td>21.6</td> | + | <td class="row">21.6</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td>E40-2</td> | + | <td class="row">E40-2</td> |
- | <td>19.9</td> | + | <td class="row">19.9</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td>E42-1</td> | + | <td class="row">E42-1</td> |
- | <td>25.1</td> | + | <td class="row">25.1</td> |
</tr> | </tr> | ||
Line 130: | Line 130: | ||
<center> | <center> | ||
- | <table | + | <table class="data"> |
<tr> | <tr> | ||
- | <td><b>DNA (μl)</b></td> | + | <td class="row"><b>DNA (μl)</b></td> |
- | <td><b>H<small><sub>2</sub></small>O (μl)</b></td> | + | <td class="row"><b>H<small><sub>2</sub></small>O (μl)</b></td> |
- | <td><b>Enzyme 1 (μl)</b></td> | + | <td class="row"><b>Enzyme 1 (μl)</b></td> |
- | <td><b>Enzyme 2 (μl)</b></td> | + | <td class="row"><b>Enzyme 2 (μl)</b></td> |
- | <td><b>Buffer H (μl)</b></td> | + | <td class="row"><b>Buffer H (μl)</b></td> |
- | <td><b>Final Volume (μl)</b></td> | + | <td class="row"><b>Final Volume (μl)</b></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td>5</td> | + | <td class="row">5</td> |
- | <td>16.5</td> | + | <td class="row">16.5</td> |
- | <td>0.5 EcoRI</td> | + | <td class="row">0.5 EcoRI</td> |
- | <td>0.5 PstI</td> | + | <td class="row">0.5 PstI</td> |
- | <td>2.5</td> | + | <td class="row">2.5</td> |
- | <td>25</td> | + | <td class="row">25</td> |
</tr> | </tr> | ||
Line 167: | Line 167: | ||
<center> | <center> | ||
- | <table | + | <table class="data"> |
<tr> | <tr> | ||
- | <td><b>Ligation Name</b></td> | + | <td class="row"><b>Ligation Name</b></td> |
- | <td><b>Vector</b></td> | + | <td class="row"><b>Vector</b></td> |
- | <td><b>Vector volume (μl)</b></td> | + | <td class="row"><b>Vector volume (μl)</b></td> |
- | <td><b>Insert</b></td> | + | <td class="row"><b>Insert</b></td> |
- | <td><b>Insert volume (μl)</b></td> | + | <td class="row"><b>Insert volume (μl)</b></td> |
- | <td><b>Buffer (μl)</b></td> | + | <td class="row"><b>Buffer (μl)</b></td> |
- | <td><b>T4 Ligase (μl)</b></td> | + | <td class="row"><b>T4 Ligase (μl)</b></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td><b>E41N</b></td> | + | <td class="row"><b>E41N</b></td> |
- | <td>E36 (S-P)</td> | + | <td class="row">E36 (S-P)</td> |
- | <td>4</td> | + | <td class="row">4</td> |
- | <td>E3-1 (X-P)</td> | + | <td class="row">E3-1 (X-P)</td> |
- | <td>4</td> | + | <td class="row">4</td> |
- | <td>1</td> | + | <td class="row">1</td> |
- | <td>1</td> | + | <td class="row">1</td> |
</tr> | </tr> | ||
</table> | </table> | ||
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<center> | <center> | ||
- | <table | + | <table class="data"> |
<tr> | <tr> | ||
- | <td></td> | + | <td class="row"></td> |
- | <td><b>E37</b></td> | + | <td class="row"><b>E37</b></td> |
- | <td><b>E38</b></td> | + | <td class="row"><b>E38</b></td> |
- | <td><b>E39</b></td> | + | <td class="row"><b>E39</b></td> |
- | <td><b>E40</b></td> | + | <td class="row"><b>E40</b></td> |
- | <td><b>ENTERO4C5</b></td> | + | <td class="row"><b>ENTERO4C5</b></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td><b>t = 0 h</b></td> | + | <td class="row"><b>t = 0 h</b></td> |
- | <td>0.12</td> | + | <td class="row">0.12</td> |
- | <td>0.11</td> | + | <td class="row">0.11</td> |
- | <td>0.13</td> | + | <td class="row">0.13</td> |
- | <td>0.10</td> | + | <td class="row">0.10</td> |
- | <td>0.11</td> | + | <td class="row">0.11</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td><b>t = 1 h</b></td> | + | <td class="row"><b>t = 1 h</b></td> |
- | <td>0.22</td> | + | <td class="row">0.22</td> |
- | <td>0.15</td> | + | <td class="row">0.15</td> |
- | <td>0.21</td> | + | <td class="row">0.21</td> |
- | <td>0.28</td> | + | <td class="row">0.28</td> |
- | <td>0.19</td> | + | <td class="row">0.19</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td><b>t = 4 h</b></td> | + | <td class="row"><b>t = 4 h</b></td> |
- | <td>0.47</td> | + | <td class="row">0.47</td> |
- | <td>0.60</td> | + | <td class="row">0.60</td> |
- | <td>0.53</td> | + | <td class="row">0.53</td> |
- | <td>0.37</td> | + | <td class="row">0.37</td> |
- | <td>0.53</td> | + | <td class="row">0.53</td> |
</tr> | </tr> | ||
Revision as of 14:48, 17 August 2011